Osteoarthritis (OA) is a painful, debilitating disease associated with a significant socioeconomic burden. Many potential therapeutics for OA would likely be more effective if they could be applied at early disease stages, and to facilitate this, development of diagnostics for early stage OA is a critical need for the future of OA therapy. Unfortunately, technologies to assess joint-level changes in small articular joints are underdeveloped, and these technological limits impede our ability to effectively develop new diagnostic and prognostic biomarkers for OA. Our group has developed a new magnetic nanoparticle based technology to extract biomarkers directly from small articular joints without the need to aspirate synovial fluid. Using magnetic capture, we have demonstrated the ability to detect an OA biomarker from a rat knee with greater accuracy and sensitivity than current methods. The objective of this proposal is to advance magnetic capture as a method to assess multiple biomarkers in rodent OA models following animal euthanasia (Aim 1) and as a method to assess biomarker levels in live animals (Aim 2). These advances will help enable the development of prognostic biomarkers through rodent OA models, improve the preclinical assessment of emerging OA therapeutics, and demonstrate the long-term potential to apply magnetic capture to OA biomarker analysis in small human joints, such as those of the human hand. This application focuses on two, multi-objective specific aims. In Aim 1, magnetic capture will be advanced as post mortem analysis method of joint-level OA biomarker assessment in rodents by completing the following objectives: (1.1) Develop multiplex methods to detect multiple OA biomarkers in a single magnetic capture procedure, (1.2) Refine the magnetic probe design to increase the efficiency of the magnetic nanoparticle capture, (1.3) Demonstrate feasibility and utility of magnetic capture in a mouse OA model, and (1.4) Evaluate links between joint-level OA biomarkers, histological evidence of joint degeneration, and behavioral evidence of OA symptoms. In Aim 2, magnetic capture will be advanced as an in vivo analysis method of joint-level OA biomarker assessment in rodents by completing the following objectives: (2.1) Design particles and targeting molecules for the in vivo capture of OA biomarkers, (2.2) Design a catheter-based injection/collection apparatus for the in vivo capture of magnetic nanoparticles, (2.3) Assess the clearance, safety, and biocompatibility of magnetic particles used for in vivo magnetic capture, and (2.4) Longitudinally assess a joint-level OA biomarker in the rat using in vivo magnetic capture. Successful completion of these Aims and Objectives will lead to better understand the links between OA-related pathogenesis and OA-related pain and disability, and thereby an ability to develop diagnostic and prognostic OA biomarkers that will improve patient outcomes for OA. A major step toward this goal is the development of an enabling technology for longitudinal OA biomarker assessment in the synovial fluid of small articular joints. In this proposal, our novel magnetic capture technology will be advanced to address this need.